GSK3 inhibition reduces ECM production and prevents age-related macular degeneration–like pathology

Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) is an age-related macular degeneration–like (AMD-like) retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production in retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus that enabled simple, sensitive, and high-throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix, reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium-derived factor). In vivo, treatment of 8-month-old R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is an important demonstration of small molecule–based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of retinal degenerative diseases, including potentially AMD itself.


Expanded Methods from Main Document
Cell culture.ARPE-19 cells (CRL-2302, ATCC) were cultured in DMEM/F12 (Corning) with 10% FBS (Omega Scientific) and 1% PSQ (ThermoFisher).Cells were kept at 37°C with 5% CO2, and passages were performed every 3-4 days, or when confluent.For experiments, ARPE-19 cells were plated at confluence (unless otherwise noted), typically at a concentration of ~100,000 cells/cm 2 , which equates to ~200,000 cells/mL.HEK-293T cells (Life Technologies) were cultured in DMEM High Glucose (4.5 g/L) supplemented with 10% FBS and 1% PSQ.Mouse NIH-3T3 fibroblasts (CRL-1658, ATCC) were maintained in DMEM high-glucose with 10% calf serum and 1% PSQ.Primary dermal fibroblasts (PCS-201-012, ATCC) were maintained in either low glucose DMEM with 10% FBS and 1% PSQ, or fibroblast growth media (Fibroblast Basal Medium [PCS-201-012, ATCC] supplemented with Low-Serum Fibroblast Growth Kit [PCS-201-041, ATCC]. Cells were periodically confirmed to be free of mycoplasma contamination using the Universal Mycoplasma Detection Kit (30-1012k, ATCC).HiBiT F3 cell line generation.Low passage ARPE-19 cells were genomically edited to introduce a 2xFLAG-VS-HiBiT sequence immediately after the signal sequence cleavage site (Ser16) of F3 (Figure 1A).This area in F3 was specifically chosen because an extreme N-terminal tag would be cleaved off after co-translational import into the endoplasmic reticulum (ER), and because we have anecdotally noticed that C-terminal appendages (of any size) compromise the secretion efficiency of F3, in line with recent observations that a stop codon mutation in EFEMP1 results in intracellular retention and causes a juvenile form of glaucoma (12).Introduction of the 29 amino acid 2xFLAG-VS-HiBiT tag at this position was not predicted to affect the native F3 signal sequence cleavage (Supplemental Figure 1, A and B), and FLAG tags have been used in a similar manner to force signal sequence cleavage immediately prior to the tag (85).For an in-depth description of the editing process, please refer to a recent protocol paper (86).Briefly, a CRISPR/Cas9 ribonucleoprotein (RNP) was generated using Alt-R Sp.Cas9 Nuclease V3 (Integrated DNA Technologies, IDT) loaded with a crRNA/tracrRNA duplex (Supplemental Table 1).This RNP, combined with a single-stranded oligodeoxynucleotide (ssODN) repair template (Supplemental Table 1) were introduced into ARPE-19 cells using electroporation (1400 V, 20 ms, 2 pulses, Neon Transfection System, Life Technologies).After electroporation, cells were incubated in antibiotic-free media containing the DNA ligase IV inhibitor, SCR7 (1 M, Sigma) for 48 h to promote homology-directed repair (HDR).Heterogenous cultures were expanded, assayed for secreted HiBiT via a luciferase assay and HiBiT blotting, and verified genomically for insertion of the 2xFLAG-VS-HiBiT sequence (Figure 1B, primers listed in Supplemental Table 2).
Full, uncropped gel images are located in Supplemental Figure 12.
High-throughput screening (HTS).Heterogenous ARPE-19 edited cells were used for all screening experiments due to their growth properties in comparison to single colony clones, which generally had much slower growth, making them subpar for large-scale HTS purposes.HiBiT F3 ARPE-19 cells were seeded at a density of ~5,000 cells/well (in 30 L with a MultiFlo, BioTek) in a white 384 well plate (781098, Greiner), and incubated at 37°C for 24 h.Media was exchanged using an EL406 microplate washer (BioTek).Compounds from the Prestwick Compound Library (1,200 compounds,Prestwick) and the National Institutes of Health clinical collection (446 compounds) were added using an Echo 655 (Beckman Coulter) at a final concentration of 5 M and 0.1% DMSO.As a positive control for reduction of F3 secretion/production, brefeldin A (BFA, 50 µM) was used for the first column of each screening plate.Twenty-four hours after compound addition, plates were cooled to RT on the benchtop for 20 min followed by assaying for HiBiT signal in a whole well reading.LgBiT and lytic substrate (Promega) were added 1:100 and 1:50, respectively, into lytic buffer to generate a master mix.Fifteen microliters of the lytic/LgBiT/substrate master mix was dispensed into each plate using a MultiFlo, shaken for 5 min, and luminescence was detected on an Envision plate reader (PerkinElmer).
HiBiT assay performance was calculated by determining the Z factor (Z') using the following equation: Hit compounds were identified as having HiBiT F3 values less than three standard deviations from the mean of all compounds.A counter screen was used to identify toxic compounds that would be predicted to yield a false positive result (i.e., reduction in F3 signal due to cell death).
Cells were plated using the same methods as the initial screen.Cherry-picked hit compounds were added the following day at the standard screening concentration (5 µM) and an additional dose that was 0.5 log below the screening dose.The Cell Titer Glo 2.0 (Promega) viability assay was performed after 24 h of treatment.A whole-well analysis was completed by adding 10 µL of Cell Titer Glo diluted 1:1 with PBS + 0.1% TX-100 to the plate and shaking for 5 min.
Luminescence was again read on the Envision plate reader (PerkinElmer).
In addition to the cell viability counter screen, media alone was treated with the hit compounds to identify any false reductions in luminescent signal caused by NanoBiT luciferase inhibition.Briefly, conditioned media was collected from ARPE-19 HiBiT F3 cells, spun, and plated into 384-well plates.Hit compounds were added in a dose-response format and incubated for 24 h.Lytic buffer/LgBiT/substrate master mix was added to the plates, shaken for 5 min, then read on an Envision plate reader.Wells that had significantly lower amounts of HiBiT signal were noted and the corresponding compounds were removed from subsequent experiments.Remaining hit compounds were verified in a confirmatory screen using fresh compound from a new source plate.
Additional secondary metrics verified the effect of a hit compound in multiple assays and in different cell lines.
Short interfering RNA (siRNA).siRNA knockdown was used to verify the specificity of CRISPR HiBiT editing of F3 or fibulin-5 (F5).siRNAs (Silencer Select, Ambion, Supplemental Table 3) were introduced into cells containing HiBiT-tagged human F3, mouse F3, or human F5 by reverse transfection.For a 24 well plate, 2.14 L of DharmaFECT4 (Horizon) was diluted into 250 µL OptiMEM (Thermo Fisher Scientific).siRNAs were added at a concentration of 100 nM.All samples were vortexed for 15 sec, then incubated at RT for 20 min.During this incubation period, ARPE-19 cells were trypsinized and normalized to a density of 466,000 cells/mL in full DMEM/F12 media.Two-hundred and fifty microliters of the siRNA/OptiMEM/DharmaFECT4 complex was added to a 24-well plate, after which an equivalent volume of cell suspension was added, yielding a final siRNA concentration of 50 nM.Plates were rocked to ensure cell distribution and to prevent clumping.After 24 h, the media was changed with full DMEM/F12.Seventy-two hours post knockdown, the media was changed again, and the HiBiT levels were determined the next day (96 h post knockdown) via an extracellular and intracellular HiBiT assay.
Non-HTS HiBiT assay.Cells were plated at a high density of 200,000 cells/mL for all non-HTS HiBiT assay experiments.The following day, media containing the indicted compound was added and incubated for either 72 h or 1 week.For one-week treatment wells, media was replaced after 96 h with fresh media containing compound.After treatment, extracellular and intracellular assays were performed to determine levels of HiBiT-tagged F3 using either the Nano-Glo HiBiT Extracellular Detection System or the Nano-Glo HiBiT Lytic Detection System (Promega).For the extracellular HiBiT protocol, 25 µL of the conditioned media was reacted with an equal volume of HiBiT master mix (extracellular or lytic buffer, 1:100 LgBiT, and 1:50 extracellular or lytic substrate) in a black-bottomed 96-well plate for 5 min with shaking.For intracellular HiBiT detection, cells were washed in well with HBSS (Gibco), followed by addition of 50 µL of lytic HiBiT master mix (described above) and 5 min shaking.Forty microliters of the lysate was transferred to a black 96-well plate for a luminescent reading on a Synergy 2 (BioTek) or a GloMax (Promega).
Western blotting.Cells for western blotting were rinsed with HBSS followed by lysis in buffer containing radioimmunoprecipitation buffer (RIPA, Santa Cruz), protease inhibitor (Pierce) and benzonase (Millipore Sigma), or the Nano-Go HiBiT Lytic Detection System.Lysis buffer was added to each well at a volume of 50 µL (for a 24 well plate, for example) followed by shaking for 5-15 min, and lysate was collected into Eppendorf tubes on ice.The samples were spun at 4°C for 10 min at 21,000 x g, and the soluble supernatant was collected.For cells lysed in RIPA buffer, a bicinchoninic acid assay (BCA, Pierce Thermo Scientific) was used to quantify protein levels and normalize them to 20 g.For cells lysed in HiBiT Lytic buffer, samples were normalized by volume due to the presence of DTT, which interferes with the BCA assay.Samples were boiled in 1x reducing Laemmli buffer for 5 min, then loaded onto a 4-20% Tris-Gly SDS-PAGE gel and run at 140 V for 80 min.Proteins were transferred to 0.2 µm nitrocellulose membrane using a semidry apparatus (P0 protocol, iBlot2, Life Technologies).Total transferred protein was visualized with Ponceau S (Sigma-Aldrich) and incubated overnight in blocking buffer (Intercept Blocking Buffer, LI-COR).The following day, blots were incubated for 1 h at RT with primary antibody [GSK3 (sc-5264, Santa Cruz), GSK3 (9315S, Cell Signaling), or GAPDH (sc-47724, Santa Cruz)] diluted in 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) and 0.05% NaN3.Blots were rinsed in TBS with 0.05% Tween (TBS-T) and incubated for 40 min at RT in an appropriate anti-mouse or anti-rabbit secondary antibody (1:10,000-15,000, LI-COR) dissolved in 5% milk in TBS-T.Blots were again washed in TBS-T, then TBS, followed by water, and imaged on an Odyssey CLx (LI-COR), followed by analysis using the corresponding ImageStudio Software (LI-COR).Full, uncropped blot images are located in Supplemental Figure 12.
HiBiT blotting.Samples were lysed, prepared, and separated by SDS-PAGE as described above for western blotting.Normalized protein was reduced, denatured, and run through an SDS-PAGE gel, followed by transfer to a nitrocellulose membrane (P0 program, iBlot2).Once transferred, the membrane was incubated in TBS-T (10 min to 1 h, RT) to expose the HiBiT epitope.Blots were then incubated with blotting buffer containing 1:200 LgBiT for 1-2 h with rocking at RT (N2410, Promega).Next, NanoGlo substrate was added (1:500) and the blots were incubated for 5-10 min at RT.The resulting chemiluminescence signal was imaged on an Odyssey Fc (LI-COR).Full, uncropped blot images are located in Supplemental Figure 12.
Lactate dehydrogenase (LDH) release assay.Cells were treated with CHIR for either 72 h or 1 week.Media was then collected and spun at 1,000 RPM (100 x g) for 5 min to remove potential floating cells.Fifty microliters (technical triplicates) of cleared media was mixed with an equal amount of LDH reaction buffer (LDH Cytotoxicity Assay Kit, Pierce) in a clear 96-well plate for 30 min at 37°C.A DMSO-treated media sample served as a control while cells treated with cell lysis buffer (provided in the LDH Cytotoxicity Kit) served as a positive control.Fifty microliters of stopping solution was added to each well and the absorbance at 490 nm was read on a GloMax plate reader(Promega).
7xTCF-eGFP mCherry (7TGC) and 7xTCF-firefly luciferase puromycin (7TFP) lentivirus production.VSV-g-pseudotyped replication incompetent lentivirus was generated as we have described previously (88, 89).Briefly, lentiviral plasmids (Addgene plasmids #24304 and #24308, kind gifts of Roel Nusse (42)) were co-transfected with psPAX2 and VSV-g plasmids (Addgene plasmids #12260 and #12259, gifts of Didier Trono) into low passage HEK-293T cells plated at 1x10 6 cells/well of a poly-D-lysine-coated 6-well plate using Lipofectamine 3000 (Life Technologies).The next day, media was discarded and replaced with fresh media.Media collected 24 h and 48 h later was pooled and filtered through a 0.45 m filter.Lenti-X GoStix (Takeda) were used to confirm the production of virus.To establish 7TGC and 7TFP cell lines, HiBiT F3 ARPE-19 cells were plated at 1x10 6 cells/well of a 6 well plate and infected with lentivirus in full media containing polybrene for 24 h.7TGC cells were not put under selective pressure whereas 7TFP cells were then selected with 1 g/mL of puromycin for 1-2 weeks.
Firefly luciferase (FLuc) assay.The HiBiT F3 7TFP ARPE-19 cells were treated with CHIR for 1 week (with a 72 h media change) and assayed for FLuc expression.Briefly, after a total of 1 week of treatment, media was aspirated and cells were washed with HBSS.Fifty microliters lysis buffer (Firefly Luciferase Glow Assay, Pierce) was added to each well, then the plate was rocked for 15 min.To monitor FLuc activity, 50 µL of working solution containing luciferin and Firefly Glow Assay Buffer was added to a black 96-well plate.Ten microliters of the lysate was added to respective wells, then read on a Synergy2 plate reader.
MMP2 zymography.HiBiT WT F3 or HiBiT R345W F3 ARPE-19 cells were plated at a density of ~100,000 cells per well of a 12-well, or ~30,000 cells per well of a 24-well, 0.4 m polyester transwell plate (Corning), and media was changed to serum free media the following day and every 3-4 days thereafter.After 1 week growing on transwells, cells were treated with either DMSO (0.1%) or 1 M CHIR in serum free media.After 2 weeks on transwells (1 week of treatment), an extracellular HiBiT assay and MMP2 zymography were performed.Briefly, for zymography, media from the apical and basal chamber was collected and combined with nonreducing SDS buffer followed by running on a 10% gelatin gel (Novex) for 90 minutes at 140 V.
MMP2 was renatured in buffer (G-biosciences) for 30 min at RT. Gels were then changed to developing buffer (G-Biosciences) for an additional 30 min at RT.After changing to fresh developing buffer, gels were gently shaken overnight at 37°C.The following day, the gels were stained in Coomassie R-250 for 1 h, then destained and imaged on an Odyssey Clx (LI-COR).
Bands were quantified using Image Studio software (LI-COR).Full, uncropped gel images are located in Supplemental Figure 12.
Secreted proteome visualization and mass spectrometry.HiBiT ARPE-19 cells were treated with CHIR for 72 h in serum-free DMEM/F12 media.To visualize separated total protein, conditioned media was concentrated (Amicon Ultra 0. was used as an internal standard.Back-calculation of standard curve and quality control samples were accurate to within 15% for 85-100% of these samples at concentrations ranging from 0.5 ng/ml to 10000 ng/ml.

Additional Methods (appearing only in Supplemental Material)
Porcine retinal pigment epithelium (RPE) culture.To assess the long-term effects of CHIR on RPE tight junction formation and maintenance, primary porcine RPE cultures were used.Briefly, procured pig eyes were processed according to Toops et al 2014 Exp.Eye Res. with slight modification.Posterior eye cups were dissected and RPE were isolated by trypsinization followed by expansion in T25 culture dishes (5 eyes per dish) for 2 weeks in differentiation media (with 10 mM nicotinamide).Cells were then trypsinized and seeded on transwells at a density of 300,000 cells/cm 2 on collagen-coated (5 g/cm 2 ) 0.4 m transwells (Corning).Cells were allowed to differentiate for 2 additional weeks prior to treatment with DMSO (1:1,000) or CHIR (1 M).Cells were then washed 3x with HBSS, fixed in 4% PFA (30 min, RT), washed again with HBSS, and stained with CytoPainter phalloidin-iFluor 647 (Abcam, 1:250 in PBS, 1% BSA, 0.5% Triton-X-100, 0.5% Tween-20, 1 h, RT).Transwells were washed again, excised and mounted using Fluoromount G (ThermoFisher) on microscope slides and imaged on a Keyence BZ-X810 at 40x magnification.
HiBiT mouse F3 cell line generation.To add HiBiT to mouse F3, NIH-3T3 fibroblasts (ATCC CRL-1658) were used.Similar to the procedure described above, a 2xFLAG VS HiBiT sequence was inserted immediately after the Ser17 residue of mouse F3 (Supplemental Figure 5, A and B).
Unlike in EFEMP1, no mutation of the PAM site was required in Efemp1 due to significant disruption of the gRNA sequence after HiBiT insertion (Supplemental Figure 6A).RNP, ssODN, and electroporation enhancer were introduced into the NIH-3T3 cells (1400 V, 20 ms, 2 pulses, Neon Transfection System).After electroporation, cells were incubated in antibiotic-free media containing HDR Enhancer V3 (1 M, IDT) for 48 h to promote homology-directed repair.
Heterogenous cultures were expanded and assayed for secreted HiBiT via a luciferase assay.
HiBiT fibulin-5 (F5) cell line generation.As a control cell line for HiBiT F3 cells, we appended HiBiT onto endogenous fibulin-5 (FBLN5, DANCE, or F5), a highly homologous protein to F3 (19), in a similar manner, immediately after the F5 signal sequence cleavage after residue Ala23 (Supplemental Figure 8A).No mutation of the PAM site was required in FBLN5 due to significant disruption of the gRNA sequence after HiBiT insertion (Table S1).RNP, ssODN, and electroporation enhancer were introduced into ARPE-19 cells using electroporation (1400V, 20 ms, 2 pulses, Neon Transfection System).After electroporation, cells were incubated in antibioticfree media containing HDR Enhancer V3 (1 M, IDT) for 48 h to promote homology-directed repair.Heterogenous cultures were expanded and assayed for secreted HiBiT via a luciferase assay.CO

Figure 1 :Supplemental Figure 2 :Supplemental Figure 3 :Supplemental Figure 4 :Supplemental Figure 5 :Supplemental Figure 6 :Supplemental Figure 7 :Supplemental Figure 8 :Supplemental Figure 9 :
Introduction of the 2xFLAG-VS-HiBiT tag is not predicted to alter human F3 signal sequence cleavage.(A) SignalP5.0predicted signal sequence cleavage of human wild-type (WT) F3 and (B) human F3 after introduction of the 2xFLAG-VS-HiBiT tag.Example data from HTS plate indicating assay uniformity and identification of potential hit compounds.(A) Example plate data from a whole-well HiBiT assay mock screen performed with DMSO and BFA (as a positive control) to identify an assay Z'score.(B, C) Representative screening data identifying a potential F3 reducer (B) or a potential F3 enhancer (C).(D, E) Linearity of the HiBiT luminescence assay.Conditioned media from WT HiBiT F3 cells was diluted over a range of concentrations followed by a HiBiT assay.Regression analysis shows excellent linearity over 6 logs of dilution.n = technical triplicates performed in two independent experiments for D, E. Select hit compounds identified in the primary screen were confirmed in dose-response.(A) Seven hit compounds were tested at 0.2 nM -50 M for 24 h.All hits reproduced and demonstrated varying levels of dose-responsiveness. (B) Table of 24 h, whole-well EC50 values obtained from panel A. Verification of CHIR99021 activity toward F3 in a human non-RPE cell line and primary porcine RPE.(A) F3 in primary human dermal fibroblasts was edited with a HiBiT tag followed by treatment with CHIR99021 for 72 h.n ≥ 3 independent experiments with mean of each experiment presented as a single data point in this graph.** p < 0.01, one sample t-test vs. hypothetical mean of 1 (i.e., unchanged).(B,C) Primary porcine RPE grown on transwells were treated with DMSO (1:1,000) or CHIR (1 M) for 11 weeks followed by staining for phalloidin (F-actin) to delineate RPE cell borders.Scale bar = 100 m.Introduction of the 2xFLAG-VS-HiBiT tag is not predicted to alter mouse F3 signal sequence cleavage.(A) SignalP5.0predicted signal sequence cleavage of mouse (mus) wild-type (WT) F3 and (B) human F3 after introduction of the 2xFLAG-VS-HiBiT tag.HiBiT editing of mouse fibroblast NIH-3T3 cells followed by treatment with CHIR99021 demonstrates cross species activity of the compound.(A) Schematic demonstrating the 2xFLAG-VS-HiBiT F3 design for genomic insertion into the mouse genome.The upward arrow indicates the predicted signal sequence cleavage site based on SignalP5.0prediction.(B) The origins of the HiBiT signal were confirmed to be from mouse F3 as demonstrated by siRNA knockdown experiments.n = 3 independent experiments, **** p < 0.0001, t-test vs. non-targeting siRNA.(C) Seventy-two hour treatment with CHIR99021 reduces F3 production in mouse cells.n = 3 independent experiments, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA with Dunnett's multiple comparison test vs. vehicle (DMSO) treated samples.Introduction of the 2xFLAG-VS-HiBiT tag is not predicted to alter human fibulin-5 (F5) signal sequence cleavage.(A) SignalP5.0predicted signal sequence cleavage of human wild-type (WT) F5 and (B) human F5 after introduction of the 2xFLAG-VS-HiBiT tag.Production of HiBiT-tagged human F5 is also reduced by CHIR99021 treatment.(A) Schematic demonstrating the design of 2xFLAG-VS-HiBiT genomic insertion onto the N-terminus of human F5 in ARPE-19 cells.The origins of the resulting HiBiT signal were confirmed to be F5 gene expression-dependent using siRNA.Representative data of n = 3 independent experiments, average ± S.D. of technical triplicates.*** p < 0.001.(C) HiBiT F5-expressing ARPE-19 cells were treated with CHIR99021 for 72 h, followed by a HiBiT assay on conditioned media.n = 3 independent experiments performed in biological duplicates each time, * p < 0.05, **** p < 0.0001.(D) One-week CHIR99021 treatment also leads to reduced extracellular and intracellular F5 levels.n = 3 independent experiments performed in biological triplicate, ** p < 0.01, **** p < 0.0001.one-way ANOVA with Dunnett's multiple comparison test vs. non-targeting or vehicle (DMSO) treated samples for all panels.Low level, 1-week CHIR99021 treatment does not substantially affect the secreted proteome from ARPE-19 cells.Total concentrated secreted protein after 1 week of CHIR99021 treatment silver stained to visualize differences in overall protein abundance.Representative data of three independent experiments shown in biological duplicate.Supplemental Figure 10: Design and validation of R345W +/+ ARPE-19 knockin cells and their subsequent HiBiT editing.(A) Design of the R345W knockin strategy using CRISPR/Cas9 editing and homology-directed repair.(B) Genomic DNA from single colonies was isolated and validated to be homozygous ( +/+ ) for the R345W mutation.(C) The validated R345W +/+ clone was then edited to include the 2xFLAG-VS-HiBiT tag.Supplemental Figure 11: Not all TEM fields of view show BLamD.Some TEM fields were found to contain no BLamDs in both (A) vehicle (A) and CHIR99021-treated mice (B).Note the healthy basal infoldings in these images also.Scale bar = 2 m.Supplemental Figure 12: Full gel and blot images for the indicated figure.